Sakashi Nomura

Application of coupled oxidation reaction to electron microscopic demonstration of horseradish peroxidase: cobalt-glucose oxidase method

Since the horseradish peroxidase (HRP) method was first applied to the peripheral nervous system by Kristensson and Olsson 12 and subsequently to the central nervous system by LaVail and LaVai116, it has become one of the most common experimental methods for demonstrating neuronal connectivity in the nervous system (for review see refs. 10 and 15). Previous studies have shown that the light microscopical procedure for demonstrating HRP can also be used for electron microscopical visualization of the enzyme, not only of the enzyme transported retrogradely to neuronal somata and dendrites3,5,8,1a,14,17,19,26, 28, but also of that transported anterogradely to axon terminals2,4,s,9,17,22-za,z6, zS. In an attempt to increase the sensitivity of the HRP method and to improve the reliability of tracing neuronal connections using retrograde and anterograde transport of HRP in combination with electron microscopy, the cobalt method of Adams 1 and the coupled oxidation reaction utilized by Lundquist and Josefsson 18 for the determination of low levels of tissue peroxidase were applied to the histochemical visualization of HRP. The coupled oxidation method, utilizing a continuous supply of hydrogen peroxide formed in the glucose oxidase (GOD) reaction, has been shown to be at least I0 times more sensitive than other colorimetric peroxidase assays 18, and also to be applicable to electron microscopical demonstration of HRP 27. In the present study, the coupled oxidation method, when applied to the histochemical demonstration of HRP in combination with cobalt method 1, was found to give excellent electron microscopical pictures of the electron dense reaction product. The experiments were performed in more than 50 adult cats anesthetized with i.p. Nembutal (35 mg/kg). For observation of the retrograde transport of HRP, 0.1-0.5 /~1 of 2 5 ~ HRP (Toyobo Grade-I-C, RZ: 3.4) was injected, in different experiments, into the masticatory muscles 2z, perineal muscles H, or wall of the urinary bladder 25. For detection of HRP transported anterogradely to axon terminals, HRP was injected in a variety of brain areas such as the cerebral cortical areas 21 and the motor trigeminal nucleus23; a single injection of 0.01~3.1 ~1 of 50 % HRP dissolved in

Immunohistochemical localization of a metabotropic glutamate receptor, mGluR5, in the rat brain

A trpE-fusion protein containing a C-terminal sequence of a rat metabotropic glutamate receptor, mGluR5, was used to produce an antibody. On immunoblot, the antibody specifically reacted with mGluR5 expressed in mammalian cells and rat brain. Immunohistochemical analysis revealed intense mGluR5-like immunoreactivity (LI) in the olfactory bulb, anterior olfactory nuclei, olfactory tubercle, cerebral cortex, hippocampus, lateral septum, striatum, nucleus accumbens, inferior colliculus, and spinal trigeminal nuclei. The distribution pattern of mGluR5-LI corresponds very well with that of mGluR5 mRNA. Electron microscope analysis of the striatum revealed dense accumulation of immunoreaction products in dendrites which were often provided with asymmetrical synapses. These results suggest that mGluR5 is predominantly located in postsynaptic elements.

Impairment of hippocampal mossy fiber LTD in mice lacking mGluR2

Subtype 2 of the metabotropic glutamate receptor (mGluR2) is expressed in the presynaptic elements of hippocampal mossy fiber—CA3 synapses. Knockout mice deficient in mGluR2 showed no histological changes and no alterations in basal synaptic transmission, paired-pulse facilitation, or tetanus-induced long-term potentiation (LTP) at the mossy fiber—CA3 synapses. Long-term depression (LTD) induced by low-frequency stimulation, however, was almost fully abolished. The mutant mice performed normally in water maze learning tasks. Thus, the presynaptic mGluR2 is essential for inducing LTD at the mossy fiber—CA3 synapses, but this hippocampal LTD does not seem to be required for spatial learning.

IMMUNOHISTOCHEMICAL LOCALIZATION OF MU -OPIOID RECEPTORS IN THE CENTRAL NERVOUS SYSTEM OF THE RAT

Of the three major types of opioid receptors (μ, δ κ) in the nervous system, μ‐opioid receptor shows the highest affinity for morphine that exerts powerful effects on nociceptive, autonomic, and psychological functions. So far, at least two isoforms of μ‐opioid receptors have been cloned from rat brain. The present study attempted to examine immunohistochemically the distribution of μ‐opioid receptors in the rat central nervous system with two kinds of antibodies to recently cloned μ‐opioid receptors (MOR1 and MOR1B). One antibody recognized a specific site for MOR1, and the other bound to a common site for MOR1 and MOR1B.

Distribution of metabotropic glutamate receptor mGluR3 in the mouse CNS: differential location relative to pre- and postsynaptic sites

The metabotropic glutamate receptors (mGluRs) have distinct distribution patterns in the CNS but subtypes within group I or group III mGluRs share similar ultrastructural localization relative to neurotransmitter release sites: group I mGluRs are concentrated in an annulus surrounding the edge of the postsynaptic density, whereas group III mGluRs are concentrated in the presynaptic active zone. One of the group II subtypes, mGluR2, is expressed in both pre- and postsynaptic elements, having no close association with synapses. In order to determine if such a distribution is common to another group II subtype, mGluR3, an antibody was raised against a carboxy-terminus of mGluR3 and used for light and electron microscopic immunohistochemistry in the mouse CNS. The antibody reacted strongly with mGluR3, but it also reacted, though only weakly, with mGluR2. Therefore, to examine mGluR3-selective distribution, we used mGluR2-deficient mice as well as wild-type mice. Strong immunoreactivity for mGluR3 was found in the cerebral cortex, striatum, dentate gyrus of the hippocampus, olfactory tubercle, lateral septal nucleus, lateral and basolateral amygdaloid nuclei, and nucleus of the lateral olfactory tract. Pre-embedding immunoperoxidase and immunogold methods revealed mGluR3 labeling in both presynaptic and postsynaptic elements, and also in glial profiles. Double labeling revealed that the vast majority of mGluR3 in presynaptic elements is not closely associated with glutamate and GABA release sites in the striatum and thalamus, respectively. However, in the spines of the dentate granule cells, the highest receptor density was found in perisynaptic sites (20% of immunogold particles within 60 nm from the edge of postsynaptic membrane specialization) followed by a decreasing receptor density away from the synapses (to approximately 5% of particles per 60 nm). Furthermore, 19% of immunogold particles were located in asymmetrical postsynaptic specialization, indicating an association of mGluR3 to glutamatergic synapses. The present results indicate that the localization of mGluR3 is rather similar to that of group I mGluRs in the postsynaptic elements, suggesting a unique functional role of mGluR3 in glutamatergic neurotransmission in the CNS.

Antibodies inactivating mGluR1 metabotropic glutamate receptor block long-term depression in cultured Purkinje cells

Antibodies were raised against two distinct extracellular sequences of the rat mGluR1 metabotropic glutamate receptor expressed as bacterial fusion proteins. Both antibodies specifically reacted with mGluR1 in the rat cerebellum and inhibited the mGluR1 activity as assessed by the analysis of glutamate-stimulated inositol phosphate formation in CHO cells expressing mGluR1. Using these antibodies, we examined the role of mGluR1 in the induction of long-term depression in cultured Purkinje cells. In voltage-clamped Purkinje cells, current induced by iontophoretically applied glutamate was persistently depressed by depolarization of the Purkinje cells in conjunction with the glutamate application. The mGluR1 antibodies completely blocked the depression of glutamate-induced current. The results indicate that activation of mGluR1 is necessary for the induction of cerebellar long-term depression and that these mGluR1 antibodies can be used as selective antagonists.

Presynaptic localization of a metabotropic glutamate receptor, mGluR7, in the primary afferent neurons: an immunohistochemical study in the rat

An antibody which recognizes specifically a metabotropic glutamate receptor, mGluR7, was produced by using a trpE fusion protein containing a C-terminal sequence of rat mGluR7. Neuropil in laminae I and II of the dorsal horn of the rat, as well as many neuronal cell bodies in the dorsal root ganglion, showed mGluR7-like immunoreactivity; the immunoreactivity in neuropil was seen in axon terminals, which were filled with round synaptic vesicles and constituted axodendritic and axosomatic asymmetric synapses. The mGluR7-like immunoreactivity in laminae I and II in the dorsal horn was reduced after dorsal rhizotomy. The results indicate that some axon terminals of the primary afferent fibers to laminae I and II of the dorsal horn are provided with mGluR7.

Immunocytochemical localization of rat substance P receptor in the striatum.

A trp E fusion protein containing a C-terminal portion of the rat substance P receptor (SPR) was expressed in bacteria and used to produce an antibody. The antibody specifically reacted with SPR expressed in a mammalian cell line and rat striatum. Light and electron microscope analyses of the rat striatum revealed intense SPR-like immunoreactivity in neuronal somata and dendrites. These immunoreactive neurons constituted approximately 3% of the total population of striatal neurons; they were putative interneurons of large and medium-sized aspiny type.

Central distribution of afferent and efferent components of the glossopharyngeal nerve: An HRP study in the cat

Central distribution of efferent and afferent components of the glossopharyngeal (IX) nerve in the cat was studied by applying horseradish peroxidase (HRP) to the lesser petrosal nerve (LPN), carotid sinus nerve (CSN) or cervical trunk (CT) distal to the branching point of the CSN. After applying HRP to the LPN or CT, HRP-labeled neurons were distributed ipsilaterally in the lateral regions of the reticular formation from the caudalmost levels of the facial nerve genu to the rostral levels of the solitary nucleus. HRP-labeled axons of these neurons ran dorsomedially and then medially along the ventral borders of the medial vestibular nucleus and the facial nerve genu, to form a small genu rostrally at the regions medial to the facial genu and caudally at the medial tip of the medial vestibular nucleus. Subsequently, the labeled axons coursed ventrolaterally to exit from the brain stem with other IX nerve components. After HRP application to the CT, labeled neurons were also seen ipsilaterally within the rostral most levels of the nucleus ambiguous, and labeled fibers were traced ipsilaterally to the medial and interstitial subnuclei of the solarity nucleus, dorsolateral portions of the spinal trigeminal nucleus at the caudalmost levels of the alaminar parts, and ventrolateral regions of the medial cuneate nucleus. Afferent components of the CSN ended ipsilaterally in the medial subnucleus, and bilaterally in the commissural subnucleus of the solitary nucleus; within the medial subnucleus, the CSN terminals located more dorsolaterally than the CT terminals. No efferent components were found in the CSN.

Cellular localization of lipocalin-type prostaglandin D synthase (?-trace) in the central nervous system of the adult rat

We applied high‐resolution laser‐scanning microscopy, electron microscopy, and non‐radioactive in situ hybridization histochemistry to determine the cellular and intracellular localization of lipocalin‐type prostaglandin D synthase, the major brain‐derived protein component of cerebrospinal fluid, and its mRNA in leptomeninges, choroid plexus, and parenchyma of the adult rat brain. Both immunoreactivity and mRNA for prostaglandin D synthase were located in arachnoid barrier cells, arachnoid trabecular cells, and arachnoid pia mater cells. Furthermore, meningeal macrophages and perivascular microglial cells, identified by use of ED2 antibody, were immunopositive for prostaglandin D synthase. In the arachnoid trabecular cells, the immunoreactivity for prostaglandin D synthase was located in the nuclear envelope, Golgi apparatus, and secretory vesicles, indicating the active production and secretion of prostaglandin D synthase. In the meningeal macrophages, prostaglandin D synthase was not found around the nucleus but in lysosomes in the cytoplasm, pointing to an uptake of the protein from the cerebrospinal fluid. Furthermore, the existence of meningeal cyclooxygenase (COX) ‐1 and COX‐2 was investigated by Western blot, Northern blot, and reverse transcriptase—polymerase chain reaction (RT‐PCR), and the colocalization of COX‐2 and prostaglandin D synthase was demonstrated in virtually all cells of the leptomeninges, choroid plexus epithelial cells, and perivascular microglial cells, suggesting that these cells synthesize prostaglandin D2 actively. Alternatively, oligodendrocytes showed prostaglandin D synthase immunoreactivity without detectable COX‐2. The localization of lipocalin‐type prostaglandin D synthase in meningeal cells and its colocalization with COX‐2 provide evidence for its function as a prostaglandin D2‐producing enzyme. J. Comp. Neurol. 428:62–78, 2000.